Abstract
α-Amylases are starch endo-hydrolases, capable of cleaving the a-1,4 glycosidic bonds of starch, a vital carbon-storage molecule in plants. Previous research in monocot plants has characterised a large multi-gene family of secreted a-amylases, with some representatives also found in dicot plants. A recent study uncovered a divergent α-amylase gene, MdAMY8, expressed in cold-treated fruit of apple (Malus domestica), which along with an orthologue from potato, appeared to form a second family separate from earlier characterised genes. A third family of plant α -amylases was discovered here, via an apple gene, MdAMY1O, which encodes an α-amylase protein with a large N-terminal extension, relative to other α-amylases. The extension is found in some other carbohydrate active enzymes and shares features with known starch-binding motifs. Each family can be found in both monocot and dicot plants, as well as in gymnosperms, and is represented by a single gene in the model plant Arabidopsis thaliana, but by at least two genes in M. domestica; the number of introns and their relative positions varies between the gene families. The protein products of MdAMY8 and MdAMY1O were predicted to localise to the cytosol and the chloroplast, respectively. The N-terminal encoding region of each gene was fused to the gene for green fluorescent protein (GFP), and transformed into plant cells. Fluorescence microscopy revealed that MdAMY8-GFP was localised in the nucleus of the cell and in a small area surrounding the cell wall, thought to be the cytosol. The MdAMY10-GFP fusion was localised to chloroplasts in leaves and non-photosynthetic plastids in roots. The three genes of A. thaliana display distinctive expression patterns, the plastid-targeted form in particular showed strong diurnal regulation. Transgenic A. thaliana plants containing hairpin-RNA producing cassettes, designed to knock-down the expression levels of each gene, showed delayed development and flowering, but leaf starch degradation did not appear to be altered. Overexpression of MdAMY8 and MdAMY10 in A. thaliana also failed to affect starch accumulation, so the activity of the two proteins was explored further by expressing them in Escherichia coli. MdAMY8 was solubilised and purified by fusion to maltose binding protein (MBP), and showed activity against amylopectin in native PAGE gels, and against soluble and insoluble starch in agarose gels. The purified protein was active at high pH, and was not inactivated by high concentrations of EGT A. The full-length MdAMY10 protein was sparingly soluble and active against amylopectin in native PAGE gels, but all activity was lost upon purification of the protein. The N-terminal extension of MdAMY10 did not confer any significant starch-binding properties when fused to GFP. It appears that the three gene families have distinct roles in carbohydrate degradation in plant cells. Family one α-amylases are secreted from the cell, and are important in germination of seeds and possibly in degrading excess starch following cell pathogenesis. Family two α-amylases are localised to the cytosol and may degrade starch- and glycogen-like molecules that are proposed to buffer cellular carbohydrate metabolism. Family three α-amylases may initiate breakdown of native starch granules in the plastids of photosynthetic and/or starch storage tissues.